A central question in cell biology is to understand those molecular events that control the efficient intracellular transport of integral membrane proteins such as receptors, transporters and adhesion molecules during organelle biogenesis, organelle maintenance, and organelle quality control. These processes are essential for all eukaryotic cells.
We want to understand at the molecular level how integral membrane proteins are correctly and efficiently sorted through the endosomal network. We are interested in applying this knowledge to gain new insight into the role of the network during cellular and tissue function and organism-level physiology, and how network errors lead to numerous diseases including those associated with ageing and neurodegeneration. To achieve this our research is highly interdisciplinary covering biochemistry, molecular biology, cell biology, genetics and neuroscience.
Biochemistry of endosome multi-protein complexes
The endosomal sorting of integral membrane proteins (termed ‘cargo’) requires the formation of multi-protein ‘coat’ complexes, which orchestrate sequence-dependent cargo selection with the membrane remodelling required to form a cargo-enriched transport carrier. To identify these complexes we utilize unbiased quantitative proteomics, as exemplified by our identification and analysis of retromer and retriever complexes and the SNX-BAR membrane tubulating complexes. Biochemical and structural analysis is used to define individual protein:protein interactions and, where appropriate, we look to reconstitute coat assembly in order to achieve a full mechanistic understanding of coat function.
In vitro cell biology of endosomal cargo sorting
At its heart the endosomal network ensures the efficient sorting of numerous, functionally diverse cargo proteins. To maximize the impact and significance of our research we utilize quantitative proteomics to achieve an unbiased, global analysis of the sorting of thousands of cargo proteins in cultured neuronal and non-neuronal cells, and combine this with suppression and gene-editing techniques to establish how the perturbation of an individual sorting complex leads to the global remodelling of cargo sorting. From the ensuing systems-level analysis of the identified cargos, we have chosen to focus our research on precisely defining the role of endosomal cargo sorting in: cell polarity and adhesion during migration and invasion; nutrient sensing and the maintenance of lysosomal health; and, neuronal survival and activity, especially in age-related neurodegenerative disease.
In vivo genetic analysis of endosomal cargo sorting in Drosophila
Little is known of the functional significance of endosomal cargo sorting for organism-level physiology. We are therefore taking advantage of CRISPR gene-editing techniques to isolate mutant Drosophila lines targeting individual endosomal sorting complexes. Using these mutants, coupled with specific imaging and biochemical techniques, we are looking to define the role of endosomal cargo sorting during: the establishment and maintenance of cell and tissue polarity; cell migration and invasion; and synaptic activity and neuronal function.
CRISPR/Cas9 was used to tag the endogenous Drosophila Vps29 protein. GFP-Vps29 localises to punctate structures in larval wing imaginal discs. Expressing RNAi against vps29 in the posterior (right hand) half of the disc strongly reduces GFP-Vps29 levels. Expressing RNAi targeting vps35, another Retromer subunit, abolishes the punctate localisation of GFP-Vps29.
RPE1 cells were lentivirally transduced with GFP-only or GFP-VPS35 viral particles. For SILAC based proteomics, GFP-VPS35 cell lines were cultured in media containing amino acids of “medium” mass (R6K4). In parallel, GFP-only expressing cells were maintained in media containing amino acids of “light” mass (R0K0). Cells were then lysed and the lysates were immunoprecipitated using GFP-trap beads. The precipitates were combined and resolved with SDS-PAGE prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification. The Venn diagram shows the interactors of the retromer complex.
Retromer complex is required for endosome-to-plasma membrane recycling of hundreds of transmembrane proteins, termed cargoes. Suppression of retromer subunits and accessory proteins results in impaired cargo recycling and mis-trafficking to lysosomes. Endogenous Glut1 glucose transporter is redistributed to LAMP1 positive lysosomes in SNX27 and VPS35 deficient HeLa cells. Surface biotinylation reveals a decrease of plasma membrane levels of several cargo proteins in SNX27 and VPS35 deficient HeLa cells.